Method for assaying human thymidylate synthase and assay kit

ABSTRACT

This invention relates to a method for immunologically measuring human thymidylate synthase with an anti-human thymidylate synthase antibody, wherein as the antibody, at least an anti-human thymidylate synthase polyclonal antibody immobilized on an insoluble matrix is used; a method for evaluating sensitivity of cancer cells to a fluoropyrimidine antitumor drug from the results of the measurement by the measuring method; and a human thymidylate synthase measuring kit comprising an insoluble matrix with at least one anti-human thymidylate synthase polyclonal antibody immobilized thereon. By a simple immunological measuring method, human TS in a sample can be measured with high sensitivity.

TECHNICAL FIELD

This invention relates to an immunological method for measuring humanthymidylate synthase and also to a kit for the same.

BACKGROUND ART

Thymidylate synthase (EC2.1.1.45, hereinafter called “TS”) is an enzymethat catalyzes a reaction in which thymidylic acid is formed fromdeoxyuridylic acid, plays a role to supply thymine which is a basespecific to DNAs, and is one of principal rate-limiting enzymes for aDNA precursor supply pathway. Accordingly, its activity is known tobecome higher in normal or tumor tissues where cell growth is active.

On the other hand, fluoropyrimidine antitumor drugs such as5-fluorourasil and 5-fluorodeoxyuridine act against TS as a targetenzyme, and for example, 5-fluorodeoxyuridine changes intofluorodeoxyuridylic acid in vivo and inhibits TS. In particular,fluoropyrimidine antitumor drugs are known to show high administrationeffect and marked life prolongation effect for patients with a smallamount of TS in tumor cells but to exhibit low administration effect forpatients with TS in a large amount [“Gan to Kagaku Ryoho (Cancers andChemotherapy)”, 24(6), 705-712 (1997)]. The importance of TS istherefore high, for example, an advance measurement of the amount of TSin an excised tumor upon treatment of a tumor patient gives indicationsfor the determination of a treatment method and for the selection of anantitumor drug.

As conventional TS measuring methods, methods which involve biochemicalmeasurement of its enzyme activity are practiced primarily, including,for example, a method in which formation of dihydrofolic acid as areaction product is measured by an absorbance at a specific wavelengthand a method in which the amount of radiolabeled fluorodeoxyuridylicacid (FdUMP) (for example, [3H]FdUMP) which binds to TS is measured.

On the other hand, immunological measuring methods making use of anti-TSantibodies have also been reported. Known methods include, for example,a method in which TS is measured by using anti-TS monoclonal antibodiesM-TS-4 and M-TS-9 [Malgorzata M. Jastreboff et al., Biochemistry,1985(24), 587-592]; and a method in which TS is detected using anti-TSmonoclonal antibodies TA 102, TS 105, TS 106, TS 109, TS 110, TS 11A andTS 11B [Japanese Language Laid-Open Publication (PCT) No. HEI 6-507314].Also known are a blotting method in which various homogenized tumorcells are subjected to electrophoresis and subsequent to transferring,an anti-TS-IgG antibody and a labeled anti-IgG are used as a primaryantibody and a secondary antibody, respectively [“Gan to Kagaku Ryoho(Cancers and Chemotherapy”, 24(6), 705-712 (1997)]; a method in whichwells are coated with a standard TS antigen and a TS-containing testsample and an anti-TS antibody are then added to cause competition; anda method in which wells are coated with a TS-containing test sample, ananti-TS antibody is added and bound, and labeled anti-mouse IgG is thenreacted.

However, the above-described methods which biochemically measure theenzyme activity of TS are accompanied by many problems in that interalia the sensitivity is insufficient to measure enzyme activity of lowlevel, use of fresh samples is needed, test samples must be carefullyhandled to avoid enzymatic degradation, test samples are required inlarge amounts, and special techniques and facilities are needed if aradioactive substance is handled.

Further, the conventional immunological measurement methods are notconsidered to be sufficient in the irksomeness of procedures and theaccuracy of measurements. Described specifically, for example, themethod in which a detection is performed by blotting subsequent toelectrophoresis is accompanied by a problem in that it is poor inquantitativeness although its procedures are very complex, the method inwhich a test sample and an antibody are competitively reacted onantigen-coated wells involves a problem in that it has poor measurementsensitivity, and even the method in which a diluted solution of a testsample is coated on wells as it is involves a problem in that TS cannotnecessarily be coated in its entirety, resulting in poor reliability. Itis therefore the current situation that there is an outstanding demandfor a simple and high-accuracy method for the measurement of TS.

DISCLOSURE OF THE INVENTION

With the foregoing circumstances in view, the present inventors haveproceeded with various investigations. As a result, it has been foundthat use of an anti-human TS polyclonal antibody immobilized on aninsoluble carrier is very effective for specifically and efficientlycapturing a trace amount of human TS from a test sample which contains agreat number of components (impurities), leading to the completion ofthe present invention.

The present invention therefore provides a method for immunologicallymeasuring human thymidylate synthase with an anti-human TS antibody,wherein as the antibody, at least an anti-human TS polyclonal antibodyimmobilized on an insoluble carrier is used.

The present invention also provides a method for evaluating sensitivityof cancer cells to a fluoropyrimidine antitumor drug, which comprisesmeasuring an amount of human TS contained in a sample of the cancercells by the above-described method and determining the sensitivity fromresults of the measurement.

The present invention also provides a human TS measuring kit comprisingan insoluble carrier with at least one anti-human TS polyclonal antibodyimmobilized thereon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a standard curve of TS concentration versusabsorbance in an anti-human TS polyclonal antibody immobilizedplate-enzyme-labeled anti-human TS polyclonal antibody system.

FIG. 2 is a graph illustrating a correlation between the substratebinding method (activation method) and the measuring method according tothe present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

To obtain the anti-human TS polyclonal antibody for use in the presentinvention, human TS is used as an immunogen. As human TS, native humanTS available from the living body by extraction and purification(hereinafter called “nhTS”) can be used or, where it is desired toobtain human TS more easily in a large amount, recombinant human TS(hereinafter called “rhTS”) can also be used. rhTS is readily available,and has merits in that its use makes it possible to obtain an antibodywith uniform reactivity and also facilitates constitution of a system,measuring performance of which always remains constant, in a measuringkit. rhTS is therefore preferred.

nhTS can be obtained, for example, as will be described hereinafter.nhTS can be obtained with high purity by loading incubated cells of aTS-expressing human tissue or a tumor strain, which has been derivedfrom a human tumor or a homogenate of a tumor subcutaneouslytransplanted in a nude mouse or nude rat, onto a column while usingethyl 10-formyl-5,8-dideazafolate as a ligand and then conductingelution with a dUMP-containing buffer.

As an alternative, rhTS can also be obtained, for example, as will bedescribed hereinafter. Firstly, a plasmid, which can derive a glutathionS-transferase (GST)-TS fused protein by isopropyl-1-thio-β-D-galactoside(IPTG), is prepared by determining the DNA sequence of human TS and theninserting a translation region in a plasmid designed to produce theGST-TS fused protein. An Escherichia coli strain which has beentransfected with the plasmid is mass-incubated in the presence of IPTG,and the incubation medium is loaded onto a glutathion-agarose column.The thus-adsorbed GST-TS fused protein is eluted with a suitable buffer,and is then heated in the presence of thrombin and calcium chloride tocleave human TS and GST from each other. The resulting mixture is loadedfurther onto a GST-agarose column, whereby rhTS of high purity can beobtained.

The anti-human TS polyclonal antibody for use in the present inventioncan be obtained in a manner known per se in the art by administeringsuch human TS (for example rhTS) to a suitable mammal such as a mouse,rat, rabbit or sheep.

Owing to the use of the anti-human TS polyclonal antibody in a formimmobilized on an insoluble carrier, the present invention has madeimprovements to the problems of the conventional immunological measuringmethods, that is, the irksomeness of procedures and the low accuracy ofmeasurement, and can achieve extremely high sensitivity.

The method of the present invention for the measurement of human TS canbe applied to any measuring system insofar as it uses an anti-human TSpolyclonal antibody immobilized on insoluble carrier or, if necessary,the antibody and a different kind of anti-human TS antibody incombination. Specifically, the measuring method of the present inventioncan be suitably applied, for example, to latex agglutination,immunochromatography, the sandwich method, competitive radioimmunoassay,enzyme immunoassay and the like, especially to the sandwich method andlatex agglutination.

To apply the method of the present invention to latex agglutination, forexample, latex particles are coated with the anti-human TS polyclonalantibody, the antibody-coated latex particles so sensitized are reactedwith a test sample containing human TS, and a change in absorbance dueto resulting agglutination is then measured, whereby human TS can bequantitated.

To apply the method of the present invention to the competitive assay,for example, an anti-human TS polyclonal antibody immobilized matrix, atest sample containing human TS, and labeled human TS are competitivelyreacted, and the label activity of the labeled human TS bound to thematrix is then measured, whereby human TS can be quantitated.

To apply the method of the present invention to the sandwich method, forexample, an anti-human TS polyclonal antibody immobilized matrix and atest sample containing human TS are reacted to form a complex, thereaction medium is removed, a labeled anti-human TS antibody is nextreacted to form a sandwich-like reaction product of the anti-human TSpolyclonal antibody immobilized matrix and the labeled anti-human TSantibody, any excess portion of the labeled antibody is removed bywashing, and the quantity of the label immobilized on the matrix is thenmeasured, whereby human TS can be quantitated.

Specific examples of materials employed in the sandwich method caninclude, as a matrix, a glass tube or plate or glass wells or beads, aplastic tube or plate or plastic wells or beads, or ferrite particles.The anti-human TS polyclonal antibody is caused to bind to the matrix byknown physical adsorption or chemical bonding, and is used as anantibody immobilized matrix.

For labeling, an enzyme, a radioactive isotope, a fluorescent substanceor the like can be used. Of these, an enzyme is preferred. As thisenzyme, those having excellent stability and permitting easymeasurements of enzyme activities can be suitably used, such asperoxidase, alkaline phosphatase, glucose oxidase and galactosidase.Examples of a method for causing such an enzyme to bind to the antibodycan include binding reactions between amino groups of enzyme andantibody with a glutaraldehyde and binding reactions making use of acrosslinking agent containing a succinimido group, a maleimido group orthe like. A method in which the sugar chain of a peroxidase is oxidizedwith periodic acid and is allowed to bind to an amino group of anantibody is also preferred for its good yield and simple procedures.

As a substrate for the measurement of enzyme activity, one specific tothe enzyme is used. Human TS can be quantitated by causing color dye,fluorescence, light emission or the like to occur and then detecting itssignal by a measuring instrument. When a peroxidase is used as anenzyme, for example, use of hydrogen peroxide and orthophenylenediaminein combination causes a color dye producing reaction to occur inproportion to the amount of the enzyme, and use of hydrogen peroxide andluminol in combination causes a light emitting reaction to occur. Asignal corresponding to the amount of the enzyme can be obtained byperforming a measurement with a spectrophotometer in the case of thecolor dye producing reaction or with a luminometer in the case of thelight emitting reaction.

To apply the method of the present invention to immunochromatography, aknown material capable of developing a liquid, such as paper or thincellulose film, is used as an insoluble matrix. By developing a liquidsample containing human TS, the anti-human TS polyclonal antibody isimmobilized at a detecting position. For the immobilization, knownmethods can be used, with a method relying upon chemical bonds beingparticularly preferred. A labeled anti-human TS antibody is contained ina position onto which the liquid sample is dropped or to an intermediatepoint on a route along which the dropped liquid sample is developed.Since irreversible adsorption of the labeled anti-human TS antibody onthe insoluble matrix is not preferred, it is preferred to solidify thelabeled anti-human TS antibody on a thin film by using a high molecularsubstance (polyvinylpyrrolidone, polyethylene glycol, or a highmolecular saccharide such as dextran). Usable examples of the labelingmaterial can include, in addition to the above-described enzymes, metalcolloids such as gold colloid and platinum; latex particles colored withdyestuff (so-called color latexes); various fluorescent reagents such asfluorescein and europium chelate; and luminescent reagents such asacridinium derivatives. Metal colloids or color latexes are preferablyused as they do not require any additional sensitizing reaction andpermit visual observations.

Into the human TS in the sample dropped onto the insoluble matrix, thelabeled anti-TS antibody is first progressively bound and developed. Thelabeled anti-TS antibody is then captured and allowed to concentrate atthe position of the immobilized anti-human TS polyclonal antibody, and adegree of coloration or the like originated from the label at theposition is detected visually or by use of an optical apparatus, wherebythe human TS can be measured.

When in addition to the immobilized anti-human TS polyclonal antibody, alabeled antibody different in kind from the first-mentioned antibody isused in combination, illustrative of such an antibody are anti-human TSpolyclonal antibodies and monoclonal antibodies.

When an anti-human TS monoclonal antibody is used, the anti-human TSmonoclonal antibody can be obtained by incubating a hybridoma (forexample, mouse hybridoma), which can produce the monoclonal antibody,for example, in a suitable medium or within the peritoneal cavity of amammal (for example, mouse).

In general, this hybridoma can be prepared, for example, by subjectingspleen cells of an rhTS- or nhTS-immunized mammal or bird (for example,mouse) and myeloma cells of a mammal (for example, mouse) to cell fusionin accordance with the method originally outlined by Kohler and Milstein[see Nature, 256, 495 (1975)]. An illustrative medium suitably usablefor the incubation of the hybridoma is a media which contains bovinefetal serum, L-glutamine, L-pyruvic acid, and antibiotics (penicillin Gand streptomycin) in Dulbeccos modified Eagle's minimum essentialmedium. The incubation of the hybridoma is conducted, for example, at 5%CO₂ concentration and 37° C. for 3 days when it is performed in anincubation medium or, for example, for 14 days when it is performedwithin the peritoneal cavity of a mouse. From the incubation medium ormammalian ascitic fluid obtained as described above, the anti-human TSmonoclonal antibody can be separated and purified by a method commonlyemployed for the isolation and/or purification of proteins. Examples ofsuch a method can include salting-out with ammonium sulfate,ion-exchange column chromatography making use of an ion-exchangecellulose, molecular sieve column chromatography making use of amolecular sieve gel, affinity column chromatography making use ofpolysaccharide complexed with protein A, dialysis, and lyophilization.

Illustrative of the anti-human TS monoclonal antibody available asdescribed above are those produced by mouse hybridomas RTSMA1 (FERMBP-6404), RTSMA2 (FERM BP-6402), NTSMA1 (FERM BP-6401) and NTSMA2 (FERMP-6403) as will be described subsequently in Examples. These hybridomashave been deposited under the Budapest Treaty in National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry (address: 1-3,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-0046, JAPAN) (date oforiginal deposit: Jun. 30, 1998). The use of an anti-human TS polyclonalantibody immobilized on an insoluble matrix in combination with such amonoclonal antibody or polyclonal antibody in the present inventionmakes it possible to measure human TS with good accuracy.

By using an anti-human TS polyclonal antibody immobilized matrix andalso by selecting a labeled antibody, a substrate and a measuringinstrument in accordance with each assay system, the detection of humanTS becomes feasible. Further, a human TS measuring kit which is usefulfor the measuring method of this invention can be constituted by usingan insoluble matrix with at least one kind of anti-human TS polyclonalantibody immobilized thereon and also by suitably choosing otherelements in accordance with each assay system.

EXAMPLES

The following examples are presented to illustrate the present inventionin further detail, but it is to be understood that the present inventionis not limited thereto.

Referential Example 1

A. Preparation of rhTS

An Escherichia coli strain NM522, in which a plasmid prepared withrestriction endonuclease recognition sites MunI to HindIII of human TScDNA incorporated therein to express a fused protein of glutathioneS-transferase (GST) and human TS had been introduced, was incubatedovernight at 37° C. under shaking in LB medium (200 mL) (product of WakoPure Chemical Industries, Ltd.) in the presence of ampicillin (50μg/mL). The incubation medium was poured in 100 mL aliquots into twoErlenmeyer flasks which contained ampicillin-containing LB medium (1liter/flask). They were incubated at 25° C. for 3 hours under shaking,to which 0.6 mL aliquots of isopropyl-1-thio-β-D-galactoside (IPTG, 40mg/mL) were added respectively, followed by further incubation at 25° C.for 20 hours. Cells were collected by centrifugation and were thensuspended in a disrupting buffer (100 mL; 50 mM Tris, pH 7.5, 25%sucrose). “10% Nonidet P-40” (5 mL; surfactant, product of NACALAITESQUE INC.) and 1 M magnesium chloride (0.5 mL) were added. The cellswere disrupted by a sonicator, followed by centrifugation at 10,000 rpmfor 15 minutes. The supernatant was caused to pass (20 mL/hr) through acolumn packed with glutathione (GSH)-agarose (14 mL; product of SigmaChemical Co.). After the column was washed with a washing-buffer (100mL; 20 mM Tris, pH 7.5, 2 mM magnesium chloride, 1 mM DTT), the columnwas eluted with an eluting-buffer (50 mL; 50 mM Tris, pH 9.6, 5 mM GSH)such that the eluate was received in 3-mL aliquots in tubes. Byconfirming protein fractions in accordance with the Bradford's method,peak fractions (9 mL; protein concentration: 7 mg/mL) were obtained.They were immediately dialyzed against a washing-buffer described above(1 liter) to lower their pH back to 7.5, and thrombin (600 units) wasadded. The reaction mixture was treated at 37° C. for 2 hours in thepresence of 1 mM calcium chloride, whereby the GST-TS fused protein wascleaved at bound sites. The resulting mixture of GST and TS was againcaused to pass through a GSH-agarose column (20 mL/hour), the column waseluted with a washing-buffer, and protein fractions were confirmed bythe Bradford's method, whereby an rhTS solution (9 mL) was obtained.0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL BSA solutions (100 μL) were added to5-mL aliquots of the Bradford's solution, respectively, and theirabsorbances at 595 nm were measured to prepare a standard curve. An rhTSsolution (100 μL), which had been diluted fivefold in distilled water,was added to the Bradford's solution (5 mL) and the absorbance at 595 nmwas measured. As a result, the protein concentration of the rhTSsolution was found to be 3.5 mg/mL.

B. Preparation of nhTS

Purification of nhTS was conducted based on the method proposed by Rodeet al. [Rode et al., Biochemical Pharmacology, 29, 723 (1980)]. A humanlung cancer strain Lu-99, which had been subcutaneously transplanted todorsal regions of 50 male BALB/c-nu/nu mice, was removed to obtaintumors (50 g). Those tumors were added with 10 mM phosphate buffer (100mL; pH 7.5, 100 mM potassium chloride, 10 mM 2-mercaptoethanol) and werethen homogenized. The homogenate was centrifuged at 4° C. and 10,000 rpmfor 1 hour, and from the supernatant, a precipitate was obtained withammonium sulfate at 30-70% saturation. The precipitate was dissolved in10 mM phosphate buffer (pH 7.5, 0.1% Triton X-100, 10 mM2-mercaptoethanol, 20 μM dUMP). The resulting solution was loaded ontoan affinity column while using ethyl 10-formyl-5,8-dideazafolate as aligand. After the column was washed with 200 mM phosphate buffer (pH7.5, 0.1% Triton X-100, 10 mM 2-mercaptoethanol, 20 μM dUMP), nhTSproteins were eluted with 200 mM phosphate buffer (pH 7.5, 0.1% TritonX-100, 10 mM 2-mercaptoethanol, 20 μM dUMP) and collected (4 mL). 0.2,0.4, 0.6, 0.8 and 1.0 mg/mL BSA solutions (100 μL) were added to 5-mLaliquots of the Bradford's solution, respectively, and their absorbancesat 595 nm were measured to prepare a standard curve. An nhTS solution(100 μL) was added to the Bradford's solution (5 mL) and the absorbanceat 595 nm was measured. As a result, the protein concentration of thenhTS solution was found to be 0.3 mg/mL.

Referential Example 2

Procedures for Preparing a Polyclonal Antibody by Using rhTS as anImmunogen

To a rabbit (New Zealand White, female, 12 weeks old), the rhTS obtainedin Referential Example 1A was subcutaneously administered at a dose of100 μg/rabbit at her dorsal region. The rhTS was used in a formemulsified beforehand in Freund's complete adjuvant. To the rabbit, therhTS in a form emulsified beforehand in Freund's incomplete adjuvant wassubcutaneously injected at a dose of 100 μg/rabbit four timessuccessively at intervals of 14 days to her dorsal region. After sevendays from the last immunization, a blood sample was collected from therabbit and serum was then obtained by centrifugation. The serum was thencaused to pass through a “Protein G Sepharose 4FF” column (product ofPharmacia AB). After the column was washed with a washing-buffer (20 mMsodium phosphate, pH 7.0), the antibody was eluted with aneluting-buffer (0.1 M glycine, pH 2.7) and was immediately dialyzedagainst the washing-buffer described above, whereby the antibody waspurified as IgG. Further, a sample of the IgG fraction was loaded ontoan rhTS-bound Sepharose 4B column. After the column was washed with awashing-buffer (20 mM sodium phosphate, pH 7.0), the antibody was elutedwith the eluting-buffer (0.1 M glycine, pH 2.7) and was immediatelydialyzed against a washing-buffer.

Referential Example 3

Preparation of a Monoclonal Antibody by Using rhTS as an Immunogen

To a female BALB/c mouse (8 weeks old), the rhTS obtained in ReferentialExample 1A was intraperitoneally injected at a dose of 20 μg/mouse. TheTS protein was used in a form emulsified beforehand in Freund's completeadjuvant. To the mouse, the rhTS in a form emulsified beforehand inFreund's incomplete adjuvant was additionally and intraperitoneallyinjected at a dose of 20 μg/mouse four times successively at intervalsof 14 days. Three days before fusion, the rhTS (100 μg) inphosphate-buffered physiological saline (0.5 mL) was injected through acaudal vein. Using spleen cells (1×10³) from the immunized mouse, P3×63Ag8 variant 653 myeloma cells (2×10⁷) and as a fusing reagent, “50%(V/V) polyethylene glycol 4000” (product of Merck & Co., Inc.), thosecells were caused to fuse together in accordance with the fusing methodproposed by Galfre and Milstein [Galfre et al., Nature 266, 550 (1977)].

After the fusion, cells were suspended in HAT medium (RPMI1640 mediumcontaining 1×10⁻⁴ M hypoxanthine, 4×10⁻⁷ M aminopterin and 1.6×10⁻⁵ Mthymidine), which contained 10% bovine fetal serum, to give a cellconcentration of 1×10⁶ cells/mL. The resultant suspension was dispensedin 200 μL aliquot per well onto a 96-well microplate.

Fused cells were incubated in a CO₂ incubator (5% CO₂, 37° C.), duringwhich replacements of the medium were conducted using HAT mediumcontaining 10% bovine fetal serum so that the fused cells were allowedto proliferate. A hybridoma formed of the spleen cells and the myelomacells were screened, and was then conditioned in HT medium (RPMI1640medium containing 1×10⁻⁴ M hypoxanthine and 1.6×10⁻⁵ M thymidine) whichcontained 10% bovine fetal serum.

The antibody in the incubation supernatant of the hybridoma was detectedin accordance with ELISA by using an rhTS-sensitized microplate. Withrespect to each well which was found to be positive, cloning wasrepeated twice in accordance with the limiting dilution analysis byusing HT medium which containing 10% bovine fetal serum and 5% Bleiclone(product of Dainippon Pharmaceutical Co., Ltd.). Two kinds of cloneswhich had reactivity to the rhTS were hence chosen, and were named“RTSMA1” (FERM BP-6404) and “RTSMA2” (FERM BP-6402).

A monoclonal antibody produced by each of the clones was obtained aswill be described next. Pristane (0.5 mL; product of DainipponPharmaceutical Co., Ltd.) was intraperitoneally injected to a nudemouse. Seven days later, pristane (0.5 mL) was intraperitoneallyadministered further, and the hybridoma (1×10⁷ cells) was transplantedto the peritoneal cavity and was allowed to proliferate. After 2 to 3weeks, ascitic fluid was obtained. The ascitic fluid was caused to passthrough a “Protein G Sepharose 4FF” column (product of Pharmacia AB).After the column was washed with a washing-buffer (20 mM sodiumphosphate, pH 7.0), the antibody was eluted with the eluent (0.1 Mglycine, pH 2.7) and was immediately dialyzed against a washing-buffer.

Referential Example 4

Preparation of a Monoclonal Antibody by Using nhTS as an Immunogen

In a similar manner as in Referential Example 3 except that as animmunogen, the nhTS obtained in Referential Example B1 was used in placeof the rhTS, two kinds of clones having high reactivity to the nhTS werechosen, and were named “NTSMA1” (FERM BP-6401) and “NTSMA2” (FERMBP-6403). From these hybridomas, monoclonal antibodies were thenobtained in a similar manner as in Referential Example 3.

Referential Example 5

Preparation of Measuring Antibodies

(1) Digestion of Anti-TS Rabbit-specific Antibody with Pepsin

The rabbit-specific antibody IgG fractions, which were obtained above inReferential Example 2, were dialyzed against 50 mM acetate buffer (pH4.5). The dialyzate was added with pepsin in an amount of 2.5% based onthe amount of IgG, and a digestive reaction was conducted for 16 hoursin an incubator controlled at 37° C. After that, the digestive reactionwas terminated by raising the pH to 8 with Tris solution, and by gelfiltration making use of a “Sephacryl S-200” column, fractions of anantibody F(ab′)₂ the molecular weight of which was 100,000 were pooled.

(2) Preparation of Antibody-immobilized Matrix

The fractions of antibody F(ab′)₂, which had been obtained as describedabove, were adjusted to 2 μg/mL with 20 mM PBS (pH 7.0), and were thendispensed in 0.1 mL aliquots onto a 96-well ELISA plate. The plate wassealed and was then subjected to coating for 2 hours in an incubatorcontrolled at 37° C., whereby an antibody-immobilized matrix wasobtained. Upon use, the coated plate was washed twice with saline (wash)which contained 0.05% Tween 20, and was then employed in a reaction.

(3) Preparation of Peroxidase-labeled Antibody

Horseraddish peroxidase (2 mg; product of Toyobo Co., Ltd.) wasdissolved in distilled water (0.5 mL), and subsequent addition of 0.2 Msodium metaperiodate (0.1 mL), a reaction was conducted at roomtemperature for 20 minutes under shaking. The reaction mixture wasloaded onto a “Sephadex G-25” column [1.5 cm in diameter×12 cm,equilibrated with 1 mM acetate buffer (pH 4.2)], and brown enzymefractions eluted with the buffer were pooled. Halves (1 mg) of thepooled enzyme were added to the polyclonal antibody IgG fraction (2 mg)and the monoclonal antibody IgG fraction (2 mg) (in 50 mM carbonatebuffer (pH 9.5, 1 mL), respectively. Further, 0.1 mL aliquots of 1 Mcarbonate buffer were added, and reactions were conducted at roomtemperature for 2 hours under shaking. 0.1 mL aliquots of 4 mg/mL sodiumborohydride were added and the resulting mixtures were allowed to standat 4° C. for 1 hour, whereby the reactions were terminated. The reactionmixtures were then separately dialyzed against saline. The innerdialyzates were separately loaded onto “Sephacryl S-200” columns (2.5 cmin diameter×70 cm, equilibrated with physiological saline), andfractionated initial peaks were pooled as enzyme-labeled antibodies,respectively.

Test 1

Comparison of Antibody Combinations

A comparison was made in measurement sensitivity by combining theanti-human TS monoclonal antibody, which was obtained from the hybridoma“RTSMA1” (FERM BP6404) in Referential Example 3, with the anti-human TSpolyclonal antibody prepared in Referential Example 5(1).

The monoclonal antibody IgG (RTSMA1) and the polyclonal antibody IgG,both of which had been adjusted to 2 μg/mL with 50 mM carbonate buffer(pH 9.5), were dispensed in 0.1 mL aliquots onto 96-well ELISA plates,respectively. The plates were sealed and were then subjected to coatingfor 2 hours in an incubator controlled at 37° C., whereby two kinds ofantibody-immobilized matrixs were obtained. After those matrixs werewashed twice with a wash, serial dilutions (0.1 mL) of purified rhTS,said dilutions having been prepared with 20 mM PBS containing 0.05%Tween 20 (diluent), were dispensed onto the antibody-immobilized plates,and the plates were statically allowed to react at 37° C. for 1 hour.After the plates were washed twice with a wash, enzyme-labeledantibodies (0.1 mL), which had been prepared from monoclonal antibodyand polyclonal antibody obtained from the hybridoma “NTSMA1” (FERMBP-6401) obtained in Reference Example 4, were dispensed into the wellsand were then statically allowed to react at 37° C. for 1 hour. Afterthe wells were washed four times with a wash, 0.1 mL aliquots of 0.1 Macetate buffer (pH 5.5, color-producing solution) which contained 3mg/mL orthophenylenediamine and 0.75 mM hydrogen peroxide were added,and enzyme reactions were allowed to proceed at room temperature for 30minutes in a dark place. Finally, 0.1 mL aliquots of 1 M sulfuric acidwere added to terminate the reactions, and measurements were conductedwith the measuring wavelength of an ELISA plate reader set at 490 nm.

The results are presented in Table 1.

TABLE 1 Absorbance (490 nm) Immobilized Immobilized ImmobilizedImmobilized monoclonal monoclonal polyclonal polyclonal antibody¹⁾antibody¹⁾ antibody antibody & & & & Concentration Labeled LabeledLabeled Labeled of rhTS monoclonal polyclonal monoclonal polyclonal(ng/mL) antibody²⁾ antibody antibody²⁾ antibody 0 0.189 0.230 0.2550.244 1 0.192 0.227 0.271 0.282 10 0.176 0.232 0.473 0.478 100 0.2420.629 0.985 1.923 1000 0.928 >3.0 >3.0 >3.0 ¹⁾RTSMA1 ²⁾NTSMA1

As is apparent from Table 1, use of an immobilized monoclonal antibodyplate has a poor response to rhTS of a low concentration and cannotprovide any high-sensitivity measuring method, while use of animmobilized polyclonal antibody can bring about high sensitivityirrespective of the kind of a labeled antibody and can measure a traceamount of human TS.

Example 1 Measurement of Human TS

(1) Measurement of a Standard and Human TS in a Sample

The protein amount of the purified rhTS obtained in Referential Example1A was quantitated, and its value was recorded as a nominal value of thestandard. The standard was diluted in 20 mM PBS (diluting solution)which contained 0.05% Tween 20, and the standard so diluted was providedfor measurement. As a sample, a centrifugal supernatant of a cancertissue homogenate was diluted tenfold in the diluting solution, and thethus-diluted supernant was provided for measurement. 0.1 mL aliquots ofthe standard or sample were dispensed onto an anti-human TS polyclonalantibody immobilized plate and were statically allowed to react at 37°C. for 1 hour. After the wells were washed twice with a wash, 0.1 mLaliquots of the enzyme-labeled anti-human TS polyclonal antibody whichhad been adjusted to an antibody amount of 1 μg/mL with the dilutingsolution were dispensed into the individual wells, and were staticallyallowed to react at 37° C. for 1 hour. After the wells were washed fourtimes with a wash, 0.1 mL aliquots of 0.1 M acetate buffer (pH 5.5;color-developing solution) which contained 3 mg/mL orthophenylenediamineand 0.75 mM hydrogen peroxide were added, and an enzyme reaction wasallowed to proceed at room temperature for 30 minutes in a dark place.Finally, 0.1 mL aliquots of 1 M sulfuric acid were added to terminatethe reaction, and measurements were conducted with the measuringwavelength of an ELISA plate reader set at 490 nm.

(2) Preparation of a Standard Curve

A graph was prepared by plotting standard TS concentrations along theabscissa and the absorbances, which had been obtained above in themeasurement (1), along the ordinate. As is shown in FIG. 1, a standardcurve was obtained with the absorbance increasing with the concentrationof the standard.

(3) Correlation with a Substrate Binding Method

A substrate binding method making use of tritium-labeled FdUMP wasconducted based on the method proposed by Spears et al. [Spears et al.,Cancer Research, 42, 450 (1982)]. A tumor tissue was added with threetimes its weight of a homogenizing buffer (200 mM Tris, pH 7.4, 20 mM2-mercaptoethanol, 15 mM cytidine 5′-monophosphate, 100 mM sodiumfluoride). Subsequent to homogenization, the mixture was centrifuged at105,000×g for 1 hour to prepare a homogenate. The homogenate (50 μL) wasadded with buffer A (50 μL; 600 mM ammonium hydrogencarbonate, pH 8.0,100 mM 2-mercaptoethanol, 100 mM sodium fluoride, 15 mM cytidine5′-mono-phosphate), buffer B (25 μL; potassium phosphate, pH 7.4, 20 mM2-mercaptoethanol, 15 mM cytidine 5′-mono-phosphate, 100 mM sodiumfluoride, 2% BSA, 2 mM tetrahydrofolic acid, 16 mM sodium ascorbate, 9mM formaldehyde), and 2 μCi/mL [³H]FdUMP (50 μL). The resulting mixturewas incubated at 30° C. for 20 minutes, and the radioactivity of anacid-insoluble fraction was measured. Assuming that TS and [³H]FdUMPwere bound with each other at 1:1, the amount of TS was calculated fromthe specific activity of [³H]FdUMP.

The amounts of human TS in the samples as obtained by the substratebinding method described above under (3) were plotted along theabscissa, while the values of concentrations converted from theabsorbances of the samples, which were obtained by ELISA in the abovemeasurement (1), by using the standard curve were plotted along theordinate. As is shown in FIG. 2, a good correlation was observed.

Capability of Exploitation in Industry

As has been described above, the present invention has made it possibleto measure human TS in a sample with high sensitivity by a simpleimmunological measuring method owing to the use of an anti-human TSpolyclonal antibody immobilized on an insoluble matrix. Quantitation ofhuman TS in a sample (for example, a stomach tissue extract) by themeasuring method of the present invention permits not only determinationof the presence or absence of a cancer, confirmation of therapeuticeffect, and the like but also provision of an indication as to whichtreatment method should be chosen and whether or not administration ofan antitumor drug is permissible.

What is claimed is:
 1. A method for immunologically measuring humanthymidylate synthase, which method comprises contacting a sample to beanalyzed with at least an anti-human thymidylate synthase antibodyF(ab′)₂ fragment immobilized on an insoluble matrix.
 2. The methodaccording to claim 1, wherein said anti-human thymidylate synthaseantibody F(ab′)₂ fragment is an anti-human thymidylate polyclonalantibody F(ab′)₂ fragment immobilized on an insoluble matrix and whereinsaid insoluble matrix on which an anti-human thymidylate synthaseantibody F(ab′)₂ fragment is immobilized is additionally contacted witha labeled anti-human thymidylate synthase antibody.
 3. The methodaccording to claim 2, wherein said anti-human thymidylate synthaseantibody F(ab′)₂ fragment is obtained using a recombinant humanthymidylate synthase as an immunogen.
 4. The method according to claim1, wherein said anti-human thymidylate synthase antibody F(ab′)₂fragment is obtained using a recombinant human thymidylate synthase asan immunogen.
 5. A method for evaluating sensitivity of cancer cells toa fluoropyrimidine antitumor drug, which method comprises measuring anamount of human thymidylate synthase contained in a sample of saidcancer cells by the method claimed in any one of claims 1-4 anddetermining said sensitivity from results of said measurement.
 6. Themethod according to claim 1, in which said method for measuring humanthymidylate synthase is a measuring system selected from the groupconsisting of latex agglutination, immunochromatography,radioimmunoassay, and enzyme immunoassay.
 7. The method according toclaim 1, in which said method for measuring human thymidylate synthaseis a sandwich method.
 8. A human thymidylate synthase measuring kitcomprising an insoluble matrix with at least one anti-human thymidylatesynthase polyclonal antibody F(ab′)₂ fragment immobilized thereon.